Immunofluorescence staining for γH2AX was adapted from previously described16 (link). After co-culture in serum-free medium supplemented with SCF (20ng/ml) and TPO (10ng/ml) for 6 days, Lin−Sca-1+c-Kit+CD48−CD150+ HSCs were directly sorted on polylysine-coated slides (P0425–72AE; Sigma), 500–2000 cells per slide, incubated for 10 min, fixed with 4 % PFA for 10 min at room temperature, and permeabilised with 0.2 % Triton X-100 for 20 min at room temperature. Subsequently, the cells were blocked with 1% BSA/PBS overnight at 4 °C. Cells were then incubated with 0.125 μg (5μl of 25μg/ml) FITC-conjugated anti-phospho-H2A.X (Ser139) antibody (Clone: 2F3; Cat: 613404; BioLegend) for 2 h at 37°C. Primary antibody staining was followed by 3 washes with PBS and 10 min incubation with 0.2 % Hoechst 33342 (Sigma). All images were acquired using ZEISS AXIO examiner D1 microscope (Zeiss) with confocal scanner unit (Yokogawa), and reconstructed in three dimensions with Slide Book software (Intelligent Imaging Innovations). Image analysis was performed using the Slide Book software (Intelligent Imaging Innovations).
P0425 72ae
Manufactured by Merck Group
Sourced in Germany
The P0425–72AE is a laboratory equipment product from Merck Group. It is a precision instrument designed for various laboratory applications. The core function of this product is to provide accurate measurements and data analysis capabilities for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Fitc conjugated anti phospho h2a x ser139 antibody, by BioLegend (2 mentions)
Axio examiner d1 microscope, by Zeiss (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Bx51trf microscope, by Olympus (1 mentions)
Rm2255, by Leica (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Dig northern starter kit, by Roche (1 mentions)
3 protocols using p0425 72ae
1
Quantifying DNA Damage in Hematopoietic Stem Cells
2
Quantifying DNA Damage in Hematopoietic Stem Cells
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Immunofluorescence staining for γH2AX was adapted from previously described16 (link). After co-culture in serum-free medium supplemented with SCF (20ng/ml) and TPO (10ng/ml) for 6 days, Lin−Sca-1+c-Kit+CD48−CD150+ HSCs were directly sorted on polylysine-coated slides (P0425–72AE; Sigma), 500–2000 cells per slide, incubated for 10 min, fixed with 4 % PFA for 10 min at room temperature, and permeabilised with 0.2 % Triton X-100 for 20 min at room temperature. Subsequently, the cells were blocked with 1% BSA/PBS overnight at 4 °C. Cells were then incubated with 0.125 μg (5μl of 25μg/ml) FITC-conjugated anti-phospho-H2A.X (Ser139) antibody (Clone: 2F3; Cat: 613404; BioLegend) for 2 h at 37°C. Primary antibody staining was followed by 3 washes with PBS and 10 min incubation with 0.2 % Hoechst 33342 (Sigma). All images were acquired using ZEISS AXIO examiner D1 microscope (Zeiss) with confocal scanner unit (Yokogawa), and reconstructed in three dimensions with Slide Book software (Intelligent Imaging Innovations). Image analysis was performed using the Slide Book software (Intelligent Imaging Innovations).
3
Floral Bud Development RNA in situ Hybridization
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Floral buds of 01-20 and HE-DGMS01-20 cabbage at different developmental stages were collected, fixed in FAA solution (50% ethanol, 5% acetic acid, 3.7% formaldehyde), and stored at 4 °C overnight. The fixed floral buds were dehydrated through an ethanol solution series of 70%, 80%, 90% and 100% followed by clearing in a xylene solution series of 25%, 50%, 75% and 100% and then embedded in paraffin (Paraplast High Melt, Leica). The samples were cut into 8–10 μm sections using a microtome (RM2255, Leica, Wetzlar, Germany) and then mounted on poly-L-lysine-coated glass slides (P0425-72AE; Sigma‒Aldrich). A 200-bp cDNA fragment from Ms-cd1 was subsequently synthesized and inserted into a pGEM-T Easy vector (Promega, Madison, WI, USA). Digoxigenin (DIG)-labeled sense and antisense probes were produced from T7 and SP6 polymerase using a DIG Northern Starter Kit (12039672910, Roche) following the manufacturer’s instructions. RNA ISH with the sense and antisense probes was performed, observed under a BX-51TRF microscope (Olympus, Tokyo, Japan) and imaged with a microcolor charge-coupled device camera (UCMOS05100KPA, ToupTek Photonics, Hangzhou, China).
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